Abstract

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in a number of linear genomes of viruses, linear plasmids and mobile elements. By this mechanism, a so-called terminal protein (TP) primes replication and becomes covalently linked to the genome ends. Bam35 belongs to a group of temperate tectiviruses infecting Gram-positive bacteria, predicted to replicate their genomes by a protein-primed mechanism. Here, we characterize Bam35 replication as an alternative model of protein-priming DNA replication. First, we analyze the role of the protein encoded by the ORF4 as the TP and characterize the replication mechanism of the viral genome (TP-DNA). Indeed, full-length Bam35 TP-DNA can be replicated using only the viral TP and DNA polymerase. We also show that DNA replication priming entails the TP deoxythymidylation at conserved tyrosine 194 and that this reaction is directed by the third base of the template strand. We have also identified the TP tyrosine 172 as an essential residue for the interaction with the viral DNA polymerase. Furthermore, the genetic information of the first nucleotides of the genome can be recovered by a novel single-nucleotide jumping-back mechanism. Given the similarities between genome inverted terminal repeats and the genes encoding the replication proteins, we propose that related tectivirus genomes can be replicated by a similar mechanism.

Figures

Figure 1.

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Bam35 TP functional assay. Lanes…

Figure 1.

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Bam35 TP functional assay. Lanes 1 and 2 show 1 μg of purified…

Figure 1.

Bam35 TP functional assay. Lanes 1 and 2 show 1 μg of purified TP and YFPTP, respectively. The Bam35 TP deoxyadenylation assay (primer-independent initiation, lanes 3–8) was carried out incubating 12.5 nM B35DNAP (except in lane 4) and 34 nM of either Bam35 TP (lanes 5 and 6) or YFPTP (lanes 7 and 8). Reactions were triggered by 1 mM MnCl2 and incubated for 30 min at 37°C. After the assay, samples of lanes 6 and 8 were treated with 0.5 μg/μl of proteinase K and 0.1% of SDS for 20 min at 37°C. Finally, all reaction products were analyzed by 12% SDS-PAGE. See Materials and Methods for details.

Mapping Bam35 TP priming residue. (A) Multiple sequence alignment of Bam35 TP and related TPs. Sequences used were from putative TPs (proteins encoded by ORF4) of representative related Gram-positive tectiviruses Bam35 (NCBI ID NP_943750.1, 10), GIL16 (YP_224102.1, 47), AP50 (YP_002302516.1, 30), as well as other BLAST-retrieved orthologous sequences from NR protein database and tentatively annotated as bacterial proteins from Bacillus cereus (WP_001085581.1), Streptococcus pneumoniae (WP_050224775.1), Exiguobacerium antarticum (WP_026829749.1), Bacillus flexus (WP_025907183.1) and Brevibacillus sp. CF112 (WP_007784052.1). These bacterial proteins may correspond to TPs from uncharacterized tectivirus-like prophages or linear plasmids from Gram-positive hosts. Sequences were aligned with MUSCLE algorithm implemented in Geneious R8 software (48). The C-terminal fragment of all proteins that corresponds with the bromide cyanogen cleavage product is shadowed in blue and the tyrosine residues present in the Bam35 portion are highlighted in pink. Conserved Y172 and Y194 residues are marked with an asterisk above the sequences. (B) Determination of the nature of the Bam35 TP priming residue by alkali treatment. Control initiation reactions with Φ29 DNA polymerase and TP were performed in parallel. After the initiation reaction, samples were incubated for 6 min at 95°C in the absence or presence of 100 mM NaOH, and subsequently neutralized and analyzed by SDS-PAGE and autoradiography. (C) Mapping the Bam35 TP priming residue. The TP-AMP complexes were performed as described and afterward treated with 1.2 mM of cyanogen bromide (CNBr) and 200 mM HCl for 20 h at room temperature. Finally, the samples were neutralized and analyzed by SDS-18% polyacrylamide electrophoresis. (D) Identification of Y194 as the priming residue by TP-deoxyadenylation assays with 0.5 or 2 μl of cell-free extracts of bacterial cultures expressing the TP variants. Extracts prepared from bacteria harboring the empty plasmid (lanes 1, 2) and the wild type TP expression vector (lanes 3, 4) were also used as negative or positive controls, respectively. See Materials and Methods for details.

Figure 4.

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Functional characterization of mutants in…

Figure 4.

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Functional characterization of mutants in the Y194 priming residue. ( A ) Template-independent…

Figure 4.

Functional characterization of mutants in the Y194 priming residue. (A) Template-independent TP-deoxyadenylation products of wild type (lane 1) and increasing concentrations of Y194F and Y194A TP mutants. Reactions were carried out triggered with 1 mM MnCl2 and incubated for 30 min. (B) Comparative analysis of wild type and Y194A and Y194F mutant TPs interaction with the DNA polymerase. The reactions were triggered with 1 mM MnCl2 in the presence of the indicated TP variant and, after 2.5 min, the competitor YFPTP fusion protein was added and the samples were incubated again for 2.5 min. See Materials and Methods for details. The effect of the TP variants concentration on the relative YFPTP deoxyadenylation, from three independent experiments (mean and standard error), is shown in panel C.

Figure 5.

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Role of conserved residue Y172…

Figure 5.

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Role of conserved residue Y172 in the interaction with the DNAP. ( A…

Figure 5.

Role of conserved residue Y172 in the interaction with the DNAP. (A) Template-independent initiation products of increasing concentrations of wild type and Y172F and Y172A TP mutants. Reactions were carried out with the indicated concentrations of TP and triggered with 1 mM MnCl2. (B) Comparative analysis of wild type and Y172A and Y172F mutant TPs interaction with the DNA polymerase. Shown are mean and standard error of three independent experiments. See Materials and Methods for details. (C) TP-primed replication of 29-mer single stranded oligonucleotide template containing the Bam35 genome origin sequence, using either wild type or Y172F TPs as primer. Time-course of full-length replication, relative to the initial events. Shown are mean and standard error of three independent experiments. The inset panel shows a representative SDS-PAGE of initiation (with dTTP) and replication (with all 4 dNTPs) products primed by the wild type or Y172F TPs after 30 min of reaction.

Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent (A) or TP-DNA directed (B) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in (C). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.

Figure 7.

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Detailed mechanism of early steps…

Figure 7.

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Detailed mechanism of early steps of protein-primed replication of single-stranded oligonucleotides containing Bam35…

Figure 7.

Detailed mechanism of early steps of protein-primed replication of single-stranded oligonucleotides containing Bam35 origins sequence variants. (A) High resolution electrophoresis of TP-primed replication products of 29-mer oligonucleotides. The two alternative sequences used 3′CTCATAC… and 3′ATCATAC…, respectively, are indicated as CTC and ATC above each lane. The combination of nucleotides included in each reaction is also detailed. The length of the different partially elongated products is indicated. Shorter bands that would correspond to TP degradation products (see Figure 1, lane 1) were not considered. Reactions were carried out as detailed in Materials and Methods, and triggered with manganese. Panel B show a schematic representation of alternative replication paths for CTC and ATC templates, respectively (see text for details). For clarity, initiator dGMP is highlighted in italic font and dideoxynucleotide bases are indicated in lower case.